The utilities and outlooks of MR cholangiography (MRCP) as a non contrast and noninvasive technique

The objective of this study was to determine the metabolic fate and disposition of the antitumor camptothecine derivative irinotecan (CPT-11). Ten patients with histological proof of malignant solid tumor received 200 mg/m2 CPT-11 as a 90-min i.v. infusion, followed by a 1.5-h i.v. infusion of cisplatin (60 or 80 mg/m2). Plasma, urine, and feces were collected for 56 h and analyzed by a specific reversed-phase high-performance liquid chromatographic assay for the parent drug and all four metabolites positively identified to date: SN-38; its beta-glucuronide conjugate, SN-38 beta-glucoronide (SN-38G); 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxycamptothecine (APC); and 7-ethyl-10-[4-N-(1-piperidino)-1-amino]-carbonyloxycamptothecine (NPC). A three-exponential decline was observed in plasma for all compounds, with a clear predominance of the parent drug [25.6+/-5.71 microM x h (CPT-11) versus 15.8+/-3.51 microM x h (total metabolites)]. Total urinary excretion was 28.1+/-10.6% of the dose, with unchanged CPT-11 and SN-38G as the main excretion products. Whereas renal clearance of SN-38 was only a minor route of drug elimination, fecal concentrations of this compound were unexpectedly high (on average, 2.45% of the dose), suggestive of intestinal hydrolysis of SN-38G by bacterial beta-glucuronidase. CPT-11 and the other metabolites could also be identified from fecal extracts, with a very minor contribution overall of the cytochrome P-450-mediated compounds 7-ethyl-10-[4-N-(1-piperidino)-1-amino]-carbonyloxycamptothecine and 7-ethyl-10-[4-N-(5-aminopentanoic acid)-1-piperidino]-carbonyloxycamptothecine. Surprisingly, fecal excretion accounted for only 24.4+/-13.3% of the dose, leading to a total excretion of approximately 52%. These data indicate that half of the dose in urine and feces may constitute some further unknown nonextractable or nonfluorescent metabolites. The findings from this study should be of importance as a guide to further therapeutic evaluation of this drug.     

Intravenous administration of irinotecan elevates the blood beta-glucuronidase activity in rats

7-Ethyl-10-hydroxycamptothecin (SN-38) is the active metabolite of an anticancer drug, irinotecan (CPT-11). Severe late diarrhea is the dose-limiting toxic effect of CPT-11. This diarrhea has been examined regarding biliary excretion and deconjugation of SN-38 glucuronide by the enzyme beta-glucuronidase (beta-GL) in intestinal microflora. Prompted by the enzymological and structural similarity of CPT-11 to organophosphorus and carbamate insecticides, we studied the effect of CPT-11 on blood beta-GL activity in rats. The i.v. injection of CPT-11 in rats significantly elevated their plasma beta-GL activity (with phenolphthalein glucuronide as a substrate) at doses of 10 and 40 mg/kg, with peak activity observed 2-3 h after administration. SN-38 lactone and carboxylate had no effect on the plasma beta-GL level. The enhancement of the activity was also observed in serum using SN-38 glucuronide as a substrate. The serum beta-GL levels showed a close correlation between these substrates. The enhancement of plasma (serum) beta-GL activity is suggested to be a result of the release of beta-GL from liver microsomes. Serum and microsomal carboxylesterase were not significantly affected by CPT-11 administration.     

Phase I clinical and pharmacologic study of weekly cisplatin combined with weekly irinotecan in patients with advanced solid tumors

PURPOSE: In vitro synergy between cisplatin and irinotecan (CPT-11) has been reported. We designed a combination schedule of these agents to maximize the potential for synergistic interaction. PATIENTS AND METHODS: To maximize the opportunity for synergy, we divided the cisplatin into four consecutive weekly treatments, followed by a 2-week rest. Each dose of cisplatin was immediately followed by a dose of irinotecan. The dose of cisplatin was fixed at 30 mg/m2/wk. The initial irinotecan dose was 50 mg/m2/wk and this was escalated by 30% increments in successive cohorts of three to six patients to establish the maximum-tolerated dose (MTD). Pharmacokinetics of irinotecan and its metabolites, SN-38 and SN-38 glucuronide (SN-38G), were analyzed. RESULTS: Of 35 patients with solid tumors enrolled onto this trial, 30 were assessable for toxicity and response. The MTD for this regimen was 30 mg/m2/wk of cisplatin plus 50 mg/m2/wk of irinotecan in previously treated patients and 30 mg/m2/wk of cisplatin plus 65 mg/m2/wk of irinotecan in chemotherapy-naive patients. Neutropenia was the dose-limiting toxicity (DLT) encountered in this trial. Diarrhea was infrequent and rarely dose-limiting. Seven of 30 assessable patients achieved a partial response. No alteration in irinotecan, SN-38, or SN-38G pharmacokinetics resulted from the administration of cisplatin with irinotecan. CONCLUSION: The administration of cisplatin and irinotecan on this weekly schedule provides a practical and well-tolerated regimen that has the potential to maximize any clinical synergy between the two agents. Evidence of substantial clinical activity was seen in this phase I study.     

Plasma pharmacokinetics of 7-ethyl-10-hydroxycamptothecin (SN-38) after intravenous administration of SN-38 and irinotecan (CPT-11) to rats

We studied the plasma pharmacokinetics of the lactone form and the carboxylate form of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), after intravenous bolus administrations of each form of SN-38 and of CPT-11 to rats. After the SN-38 lactone injection, SN-38 lactone was predominant at first, and then the lactone to the carboxylate concentration ratio (LC ratio) was maintained from 30 to 90 min after the injection. The carboxylate was predominant throughout the period after the carboxylate dosing. Model-dependent analyses showed that the SN-38 lactone had greater plasma clearance and a greater distribution volume than the carboxylate. The CPT-11 administration resulted in a predominant plasma SN-38 lactone concentration. The contribution of the SN-38 lactone AUC to the total SN-38 AUC (57%) was independent of the dose of CPT-11. These results suggest that it is possible to estimate the SN-38 lactone concentration and AUC from the total SN-38 concentration without separate determination of the lactone and carboxylate. Our results showed that both SN-38 lactone and CPT-11 administration gave the predominant SN-38 lactone in plasma; however, only CPT-11 could sustain the lactone concentration at a high level, which is necessary for antitumor activity.     

The evaluation of eustachian tube function in patients with chronic otitis media

To assess the clinical usefulness of salivary monitoring of irinotecan (CPT-11) and its active metabolite (SN-38), we examined the clinical pharmacological profile of both drugs in 9 patients with thoracic malignancies who received 60 mg/m2 CPT-11 (21 courses). Plasma and unstimulated whole saliva were collected over a 24-h period, and concentrations of CPT-11 and SN-38 were measured by high-performance liquid chromatography. Both CPT-11 and SN-38 were detectable in saliva, and the concentration-time curves in plasma and saliva showed a very similar pattern. A good correlation was observed between the saliva concentration (C3) and the plasma concentration (Cp) for both CPT-11 and SN-38 (r = 0.732, P < 0.0001 and r = 0.611, P < 0.0001, respectively). The area under the concentration-time curve calculated for saliva (AUCs) correlated with that generated for plasma (AUCp) for both CPT-11 and SN-38 (r = 0.531, P = 0.012 and r = 0.611, P = 0.0025, respectively). These results suggest that it may be feasible to use saliva instead of plasma for pharmacokinetics/pharmacodynamics studies of CPT-11.     

Pharmacokinetic characteristics of the novel anticancer agent CPT-11 and its active metabolite in plasma and lung lymph fluid following intravenous administration to sheep

7-Ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin (CPT-11, 100286-90-6) is one of the most promising novel anticancer agents, especially for lung cancer. 7-Ethyl-10-hydroxycamptothecin (SN-38), an active metabolic of CPT-11, has much stronger cytotoxicity than CPT-11. The present study was designed to evaluate the distribution and behavior of CPT-11 and SN-38 in lung lymph circulation following intravenous infusion. Awake sheep with chronically instrumented lung lymph fistulas were prepared. The concentrations of CPT-11 and SN-38 in plasma and lung lymph fluid were measured after intravenous infusion of 100 mg/body of CPT-11 for 90 min. SN-38 constantly showed higher lymph to plasma concentration ratios than those of CPT-11, and the % area under the curve (AUC) ratio of SN-38/CPT-11 in lymph fluid was significantly higher than that in plasma. These data indicated that SN-38 distributed in lung tissue moved more easily into lung lymph fluid than CPT-11, and might be more rapidly metabolized in lung tissue than plasma. CPT-11 may have favorable therapeutic effects on intrathoracic malignancies such as lung cancer and lymph metastasis.     

Unexpected hepatotoxicities in patients with non-Hodgkin's lymphoma treated with irinotecan (CPT-11) and etoposide

BACKGROUND: Irinotecan (CPT-11) is a topoisomerase I inhibitor that has been confirmed to be active against a broad spectrum of neoplasms including non-Hodgkin's lymphoma (NHL). Because the combination of topoisomerase I and II inhibitors seemed to be an attractive therapeutic strategy owing to their complementary functions, we conducted a combination phase I study of CPT-11 and etoposide, a topoisomerase II inhibitor, in relapsed or refractory non-Hodgkin's lymphoma (NHL). METHODS: The starting doses of CPT-11 and etoposide were 30 mg/m2/day (days 1-3 and 8-10) and 40 mg/m2 (days 1-3), respectively. RESULTS: All three patients who received the starting dose developed dose-limiting toxicities including one case of grade 4 neutropenia lasting for > 7 days, one of grade 3 serum transaminase elevation and one of grade 3 hyperbilirubinemia. All three patients presented hepatotoxicity > or = grade 2. The starting dose level was judged to be the maximum tolerated dose (MTD) and further dose escalation of this combination was halted. The patient who developed grade 3 hyperbilirubinemia presented a second peak of plasma SN-38, an active metabolite of CPT-11, on the concentration-time curve for day 3, suggesting the possibility of the enterohepatic circulation of SN-38 and of a drug-to-drug interaction. No durable objective response was observed in the three patients treated at the starting dose. CONCLUSIONS: We conclude that etoposide is not recommended for combination with CPT-11 in NHL patients because of unexpected frequent hepatotoxicities.     

A phase II study of irinotecan alternated with five days bolus of 5-fluorouracil and leucovorin in first-line chemotherapy of metastatic colorectal cancer

PURPOSE: This multicenter phase II study was designed to assess the efficacy of the alternating schedule of irinotecan (CPT-11) with bolus 5-fluorouracil (5-FU) and leucovorin (LV) in first-line chemotherapy for metastatic colorectal cancer (CRC). PATIENTS AND METHODS: Patients with histologically proven metastatic colorectal cancer, and at least one bidimensionally measurable lesion, aged 18-70, with performance status < or = 2, normal baseline biological values and no prior chemotherapy (or only adjuvant chemotherapy completed > or = 6 months before study entry) were selected. Treatment was irinotecan 350 mg/m2, i.v., day 1, alternating with leucovorin 20 mg/m2 i.v. and 5-FU 425 mg/m2, i.v. daily for five consecutive days, day 22-26 (Mayo Clinic regimen). One alternating cycle was to be performed every six weeks. Patients were evaluated for efficacy every alternating cycle. Treatment was administered until five alternating cycles, disease progression, unacceptable toxicity or patient refusal. RESULTS: Thirty-three patients (28 chemotherapy-na?ve and five with prior adjuvant treatment completed > 1 year prior to accrual) were enrolled. The objective response rate (RR) was 30% (95% CI: 16-49; 10 patients/33; nine partial response and one complete response). All responses were reviewed by an independent external review committee. An additional 49% of patients had stable disease. The median survival was 16 months, the one year survival amounted to 58% and the median progression free survival was 7.2 months. Relative dose intensity was nearly 90% for both drugs. Grade 3-4 diarrhea and neutropenia were the most frequent severe toxic events, seen in 24% and 64% of patients, respectively. CONCLUSIONS: The alternating schedule of CPT-11 350 mg/m2 with five days bolus of 5-FU and low dose LV is an active and feasible regimen as front-line therapy for metastatic CRC. It is well tolerated, without evidence of overlapping toxicity. The response rate appears promising with regard to that expected with either single agent. This regimen warrants further assessment in randomized trials.     

Irinotecan (CPT-11) metabolites in human bile and urine

A female patient was treated with irinotecan (CPT-11) for liver metastatic colon carcinoma. She had a percutaneous biliary catheter because of extrahepatic biliary obstruction. The patient was treated with CPT-11 for three courses at doses of 350 mg/m2 for the first course and 300 mg/m2 for the remaining courses, given as a 30-min i. v. infusion. Metabolism studies in bile and urine were performed by coupling high-performance liquid chromatography to electrospray mass spectrometry. Conventional spectra [liquid chromatography/mass spectrometry (LC/MS)] allowed on-line molecular mass determination of CPT-11 and its main metabolites, whereas structural information was obtained by tandem mass spectrometry (LC/MS/MS). At least 16 metabolites were detected in bile, while 8 of them were also detected in urine. Three compounds were identified as the parent drug, the active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38), and SN-38 glucuronide. The major metabolic pathway consists in oxidations of the terminal piperidine ring of the CPT-11 side chain, which eventually results in the formation of a primary amine. Other metabolites result from oxidation of the camptothecin nucleus. Finally, decarboxylation of the carboxylate form of CPT-11 was also observed. Several metabolites result from combinations of these pathways. The structures of the identified metabolites indicate for the first time a major role of monooxygenases in the elimination of a camptothecin derivative in humans. This finding will allow better understanding of interindividual variability in pharmacokinetics and intestinal toxicity of CPT-11.     

Simultaneous determination of the lactone and carboxylate forms of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of irinotecan (CPT-11), in rat plasma by high performance liquid chromatography

We established a high performance liquid chromatography (HPLC) method for the simultaneous determination of the lactone and carboxylate forms of 7-ethyl-10-hydroxycamptothecin (SN-38), the active metabolite of the antitumor drug irinotecan (CPT-11), in rat plasma. Plasma samples were pretreated with chilled MeOH and zinc sulfate to precipitate protein, and were then directly injected into the HPLC system. Chromatography was carried out with a Puresil C18 column (particle size 5 microns), and the mobile phase consisted of 0.1 M ammonium acetate buffer (pH 5.5) and acetonitrile (70/30, v/v) containing 20 mM of tetra-n-pentylammonium bromide. The column effluent was monitored with a spectrofluorometer (excitation wavelength 380 nm, emission wavelength 540 nm). The method was valid for SN-38 lactone (5-2500 ng/ml) and carboxylate (5-1000 ng/ml).     

A "pre-satiety sequence" in rats drinking sucrose solutions

A reversed-phase high-performance liquid chromatographic method with fluorescence detection was developed and validated for the quantitation of SN-38, the active metabolite of irinotecan (CPT-11), a new anticancer drug. This method uses solid-phase extraction with a C18 column for sample clean-up and concentration following acidification of human plasma with two volumes of 0.1 M HCl. Using blank plasma spiked with SN-38, we found the assay to be linear over the concentration range of 10-500 pM (3.9-195 pg/ml) with acceptable total and within-day imprecision. The recovery of SN-38 ranged from 48.3% (10 pM) to 91.5% (500 pM) whereas that of the internal standard, 20-(S)-camptothecin, was 96.9% (500 pM). This method represents a sizeable increase in sensitivity over other published methods and is shown to be suitable for the measurement of 'trough' concentrations of SN-38 during the treatment of patients with a weekly regimen of irinotecan.     

Purification and characterization of human sarcomeric mitochondrial creatine kinase

PURPOSE: A dose-escalation study of irinotecan hydrochloride (CPT-11) combined with fixed-dose cisplatin was conducted to determine the maximum-tolerated dose (MTD), dose-limiting toxicities, and objective response rate in patients with advanced gastric cancer. PATIENTS AND METHODS: Twenty-four patients with or without prior chemotherapy were enrolled. All patients were assessable for toxicities and response. On day 1, CPT-11 was administered as a 90-minute intravenous (I.V.) infusion, which was followed 2 hours later by a 120-minute I.V. infusion of cisplatin 80 mg/m2. CPT-11 alone at the same dose was administered again on day 15. The treatment was repeated every 4 weeks until disease progression was observed. The initial dose of CPT-11 was 60 mg/m2, and was escalated in increments of 10 mg/m2 until severe or life-threatening toxicity was observed. RESULTS: The MTD of this combination was CPT-11 80 mg/m2. At this dose level, 16.7% of patients (two of 12) had leukopenia of less than 1,000/microL, 66.7% (eight of 12) had neutropenia of less than 500/microL, and 16.7% (two of 12) had severe diarrhea of grade 4 during the first course. The dose-limiting toxicity was neutropenia. Ten patients achieved a partial response (PR), and the overall response rate was 41.7% among 24 patients (95% confidence interval, 21.9% to 61.4%). CONCLUSION: The recommended dose and schedule is CPT-11 70 mg/m2 on days 1 and 15 and cisplatin 80 mg/m2 on day 1 every 4 weeks. This combination of CPT-11 and cisplatin, considered to be active against advanced gastric cancer with acceptable toxicity, should be further assessed in a phase II study.     

Interleukin 15 offers selective protection from irinotecan-induced intestinal toxicity in a preclinical animal model

Irinotecan (CPT-11) is a chemotherapeutic agent that is active in the treatment of a variety of solid tumor malignancies. Diarrhea represents the most common dose-limiting toxicity that is independent of the schedule of administration. A rat model with dose-limiting toxicity profiles that are similar to those observed in patients treated with CPT-11 was developed and used to evaluate the role of interleukin 15 (IL-15) in the modulation of the therapeutic selectivity of CPT-11 in normal rats and rats bearing advanced colorectal cancer. The maximum tolerated dose and lethal dose (LD) of CPT-11 by i.v. push daily x 3 were 150 and 200 mg/kg/day, respectively. CPT-11 at the LD induced a 93-100% incidence of severe diarrhea and an 86-100% incidence of lethality in treated animals. IL-15, a cytokine with multiple mechanisms of action, was used at a 100 or 400 microg/kg/dose with different schedules of administration (3, 8, and 11 doses, i.p.) to protect against CPT-11-induced toxicity. IL-15 offered complete and sustained selective protection against CPT-11-induced delayed diarrhea and lethality. IL-15 also moderately potentiated the antitumor activity of CPT-11 in rats bearing advanced colorectal cancer. Morphological examination of rat intestinal tissues after treatment with LD of CPT-11 revealed dramatic protection of duodenal and colonic tissue architecture by IL-15. CPT-11 alone produced serious damage to duodenal villi and colonic crypts. The results clearly demonstrated the ability of IL-15 to provide significant protection from CPT-11-induced intestinal toxicity with maintenance of antitumor activity, resulting in an increase in the therapeutic index of CPT-11. The clinical relevance of the results obtained in this model system needs to be confirmed.     

Multispecific organic anion transporter is responsible for the biliary excretion of the camptothecin derivative irinotecan and its metabolites in rats

Irinotecan, 7-ethyl-10-[4-(1-piperidino)-1-piperidino]-carbonyloxycamptothecin (CPT-11), is a potent anticancer drug that is increasingly used in chemotherapy. A frequent limiting side effect involves gastrointestinal toxicity (diarrhea), which is thought to be related to the biliary excretion of CPT-11 and its metabolites. Accordingly, the biliary excretion mechanisms for both the lactone and carboxylate forms of CPT-11 and its metabolites, SN-38 and its glucuronide (SN38-Glu), were investigated using Sprague-Dawley (SD) rats and Eisai hyperbilirubinemic rats (EHBR), with the latter being mutant rats with a genetic deficiency of the canalicular multispecific organic anion transporter. After i.v. administration of CPT-11, the biliary excretion clearance, defined as the biliary excretion rate normalized to the hepatic concentration, of both the lactone and carboxylate forms of SN38-Glu was much lower in EHBR. The biliary excretion clearance for the carboxylate form of both CPT-11 and SN-38 was also substantially smaller in EHBR and showed marked saturation with increasing dose only in SD rats. On the other hand, the biliary excretion clearance for the lactone forms of CPT-11 and SN-38 showed only a minimal difference in EHBR, compared with SD rats. These results suggest that, for the carboxylate form of CPT-11 and SN-38 and the carboxylate and lactone forms of SN38-Glu, there exists a specific transport system at the bile canalicular membrane that is deficient in EHBR. To confirm this hypothesis, the uptake of these substrates by isolated hepatic canalicular membrane vesicles (CMV) was examined. ATP-dependence was clearly observed for the uptake of these four compounds by CMV prepared from SD rats but not by CMV from EHBR. In addition, the compounds inhibited the ATP-dependent uptake of S-(2,4-dinitrophenyl) glutathione by CMV from SD rats, in a concentration-dependent manner. These results suggest that the biliary excretion of the carboxylate forms of CPT-11 and SN-38 and the carboxylate and lactone forms of SN38-Glu is mediated by the multispecific organic anion transporter, which is deficient in EHBR.     

Activity of fotemustine in medulloblastoma and malignant glioma xenografts in relation to O6-alkylguanine-DNA alkyltransferase and alkylpurine-DNA N-glycosylase activity

Fotemustine is a chloroethylnitrosourea with antitumor activity in disseminated melanoma and adult primary brain tumors. Because new drugs are required for the treatment of medulloblastoma in children, we evaluated the preclinical antitumor activity of fotemustine in four s.c. medulloblastoma xenografts, in comparison with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU). Both drugs were administered as a single i.p. injection to nude mice bearing advanced-stage tumor. Fotemustine displayed significant antitumor activity in three of four medulloblastoma xenografts; two, IGRM34 and IGRM57, were highly sensitive, with 37 and 100% tumor-free survivors, respectively, more than 120 days after treatment at the highest nontoxic dose (50 mg/kg). Fotemustine was also highly active in a malignant glioma xenograft (IGRG88; five of six tumor-free survivors on day 177). Fotemustine proved to be significantly more active than BCNU in IGRM34 and the glioma xenograft IGRG88. The DNA repair protein O6-alkylguanine-DNA alkyltransferase (ATase) was detected in all tumor xenografts, ranging in activity from 6 to 892 fmol/mg protein. The high in vivo sensitivity to fotemustine and BCNU observed in three xenografts was clearly associated with a low ATase activity (> 20 fmol/mg), whereas the two poorly sensitive or refractory medulloblastoma xenografts showed high ATase activity (> 500 fmol/mg). Alkylpurine-DNA N-glycosylase activity was detected in all tumor xenografts but at levels ranging only from 513 to 1105 fmol/mg/h; no consistent relationship was found between alkylpurine-DNA N-glycosylase activity and the in vivo sensitivity to the two chloroethylnitrosoureas. The improved activity and tolerance of fotemustine in comparison with BCNU in pediatric medulloblastoma xenografts strongly support the clinical development of this agent in children with brain tumors, in which ATase should be examined as a potential prognostic indicator.     

The relationship between AIDS dementia complex and the presence of macrophage tropic and non-syncytium inducing isolates of human immunodeficiency virus type 1 in the cerebrospinal fluid

CPT-11 is a camptothecin derivative with a broad spectrum of antitumor activity, both in vitro and in vivo. Like camptothecin, CPT-11 is a selective DNA topoisomerase I inhibitor. Phase I trials were conducted in Europe to determine the dose and schedule for phase II trials. These phase I trials assessed the toxicity of CPT-11 in 235 patients and tested three administration schedules: a single infusion once every 3 weeks; a weekly infusion for 3 out of 4 weeks; and a daily infusion for 3 consecutive days every 3 weeks. The maximum tolerated dose (MTD) was 115 mg/m2 in the daily schedule and 145 mg/m2 in the weekly schedule. When the drug was administered once every 3 weeks, diarrhea became the dose-limiting toxicity at doses above 350 mg/m2. This schedule offered the highest dose intensity and the best tolerability profile, and was the most convenient for outpatient treatment. Finally, using this schedule, concomitant administration of high-dose loperamide allowed the dose of CPT-11 to be increased to 750 mg/m2. An ongoing phase I trial is investigating the combination of CPT-11 and 5-fluorouracil (5-FU) in various solid tumors. Although the MTD has not yet been reached, preliminary results show no pharmacokinetic interaction between the two drugs, contrary to a previous Japanese study. Based on the results of the three phase I trials, CPT-11 350 mg/m2 as an intravenous infusion over 30 minutes once every 3 weeks was recommended for phase II trials, which started in Europe in 1992. To date, CPT-11 has shown remarkable efficacy in colorectal cancer, even in patients resistant to 5-FU. Interesting results have also been obtained in pancreatic, cervical and lung cancers. Future trials will explore the place of CPT-11 in combination with other cytotoxic agents or radiotherapy.     

Active efflux of CPT-11 and its metabolites in human KB-derived cell lines

To investigate the possible involvement of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), and/or other glutathione S-conjugate export pump (GS-X pump) family members on the active efflux of irinotecan [(7-ethyl-10-[4-(1-piperidino)-1-pipertidino)-1-piperidino]carb onylox y camptothecin (CPT-11)] and its metabolites, as well as their contribution to the acquisition of resistance, we studied the uptake of CPT-11, its active metabolite SN-38, and glucuronide conjugate (SN38-Glu) using membrane vesicles from human epidermoid KB-3-1-derived cell lines. These lines included KB-C2, C-A500, and KCP-4, which overexpress P-gp, MRP, and the unidentified GS-X pump, respectively. The carboxylate form of SN-38 exhibited significant ATP-dependent transport, with a Michaelis constant of 17 microM, into membrane vesicles from C-A500 but not from other cell lines. Among these KB-derived cells, significant ATP-dependent uptake of the carboxylate form of CPT-11 was only observed in KB-C2 vesicles. In addition, the uptake of the lactone and carboxylate forms of SN38-Glu into membrane vesicles from C-A500 and KB-C2, but not KCP-4, was ATP dependent, although the transport activity in C-A500 was much higher than that in KB-C2. The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay revealed that the resistance of KB-C2 to CPT-11 and SN-38, compared with that of KB-3-1, was 6.3- and 6.8-fold, respectively; the corresponding figures for C-A500 were 12- and 27-fold, respectively, whereas those for KCP-4 were 2.3- and 20-fold, respectively. These results suggest that MRP and P-gp are involved in the active efflux of SN-38 and CPT-11, respectively, from human KB-derived cells. In addition, a difference in substrate specificity among GS-X pump members was demonstrated.     

Risperidone inhibits 5-hydroxytryptaminergic neuronal activity in the dorsal raphe nucleus by local release of 5-hydroxytryptamine

1. The effects of risperidone on brain 5-hydroxytryptamine (5-HT) neuronal functions were investigated and compared with other antipsychotic drugs and selective receptor antagonists by use of single cell recording and microdialysis in the dorsal raphe nucleus (DRN). 2. Administration of risperidone (25-400 micrograms kg-1, i.v.) dose-dependently decreased 5-HT cell firing in the DRN, similar to the antipsychotic drug clozapine (0.25-4.0 mg kg-1, i.v.), the putative antipsychotic drug amperozide (0.5-8.0 mg kg-1, i.v.) and the selective alpha 1-adrenoceptor antagonist prazosin (50-400 micrograms kg-1, i.v.). 3. The selective alpha 2-adrenoceptor antagonist idazoxan (10-80 micrograms kg-1, i.v.), in contrast, increased the firing rate of 5-HT neurones in the DRN, whereas the D2 and 5-HT2A receptor antagonists raclopride (25-200 micrograms kg-1, i.v.) and MDL 100,907 (50-400 micrograms kg-1, i.v.), respectively, were without effect. Thus, the alpha 1-adrenoceptor antagonistic action of the antipsychotic drugs might, at least partly, cause the decrease in DRN 5-HT cell firing. 4. Pretreatment with the selective 5-HT1A receptor antagonist WAY 100,635 (5.0 micrograms kg-1, i.v.), a drug previously shown to antagonize effectively the inhibition of 5-HT cells induced by risperidone, failed to prevent the prazosin-induced decrease in 5-HT cell firing. This finding argues against the notion that alpha 1-adrenoceptor antagonism is the sole mechanism underlying the inhibitory effect of risperidone on the DRN cells. 5. The inhibitory effect of risperidone on 5-HT cell firing in the DRN was significantly attenuated in rats pretreated with the 5-HT depletor PCPA (p-chlorophenylalanine; 300 mg kg-1, i.p., day-1 for 3 consecutive days) in comparison with drug naive animals. 6. Administration of risperidone (2.0 mg kg-1, s.c.) significantly enhanced 5-HT output in the DRN. 7. Consequently, the reduction in 5-HT cell firing by risperidone appears to be related to increased availability of 5-HT in the somatodendritic region of the neurones leading to an enhanced 5-HT1A autoreceptor activation and, in turn, to inhibition of firing, and is probably only to a minor extent caused by its alpha 1-adrenoceptor antagonistic action.     

Role of nitric oxide in the development of tolerance and sensitization to behavioural effects of phencyclidine in mice

1. To determine whether nitric oxide (NO) was involved in tolerance and sensitization to the effects of phencyclidine (PCP), we examined NO synthase activity and the number of NADPH-diaphorase (NADPH-d)-positive cells in discrete brain regions of saline-, acute PCP- and repeated PCP-treated mice. We also investigated the effects of a NO synthase inhibitor, NG-nitro-L- arginine methyl ester (L-NAME), on the behavioural changes induced by repeated PCP treatment in mice. 2. Acute PCP (1, 3, and 10 mg kg-1, s.c.) treatment induced dose-dependent hyperlocomotion, motor incoordination and stereotyped behaviours, consisting of sniffing, head movement and ataxia in mice. 3. In mice treated repeatedly with PCP (1, 3, and 10 mg kg-1 day-1), s.c., once a day for 14 days), the sniffing, head movement, ataxia and motor incoordination induced by PCP were attenuated (indicating the development of tolerance to these behaviours), whereas the hyperlocomotion induced by PCP was potentiated (indicating the development of sensitization to hyperlocomotion). The development of tolerance and sensitization to PCP-induced behaviours in the repeated PCP-treated mice was more marked at the dose of 10 mg kg-1 day-1) than at other doses. 4. NO synthase activity in the cerebral cortex and cerebellum, but not in the striatum and hippocampus, was significantly decreased by acute PCP (10 mg kg-1) treatment in comparison with saline treatment, and such changes in activity in the cerebral cortex and cerebellum were reversed by repeated PCP treatment (10 mg kg-1 day-1). 5. The number of neurones containing NADPH-d reactivity in the cerebral cortex, nucleus accumbens, and striatum of acute and repeated PCP-treated mice showed no change in comparison with saline-treated mice. 6. Tolerance to PCP (10 mg kg-1 day-1)-induced ataxia and motor incoordination was significantly attenuated by combined treatment with L-NAME (50 mg kg-1 day-1 i.p.). 7. Sensitization to PCP-induced hyperlocomotion was further enhanced by combined treatment with L-NAME (50 mg kg-1 day-1). However, NG-nitro-D-arginine methyl ester (D-NAME, 50 mg kg-1 day-1, i.p.), a less active enantiomer of L-NAME, had no effect, suggesting a stereospecific mechanism. 8. The PCP-induced behaviours in animals that had exhibited tolerance and sensitization to PCP (10 mg kg-1 day-1) were not influenced by acute L-NAME (5 and 50 mg kg-1, i.p.) or D-NAME (50 mg kg-1, i.p.) treatment. 9. These results suggest that NO may play an important role in the development, but not in the maintenance, of tolerance and sensitization to the effect of PCP in mice.     

Reduced plaque accumulation on hydrocarbon thin film deposited on restorative acrylic polymers

The anti-tumour activity of acriflavine in combination with guanosine has been evaluated in solid or ascitic tumour-implanted animal models. Guanosine is known to potentiate the anti-tumour effects of some chemotherapeutic agents. Administration of acriflavine (15 mg kg-1 day-1, i.m., 14 days) to ICR mice subcutaneously implanted with Ehrlich carcinoma resulted in approximately 30% inhibition in tumour growth. In contrast, minor tumour growth inhibition was observed in animals treated with guanosine at the same daily dose. Treatment of animals with both acriflavine and guanosine (AG60, 1:1, w/w) at 30 mg kg-1 resulted in approximately 65% inhibition in tumour growth rate. Whereas treatment with acriflavine or guanosine resulted in 70% or 30% decrease in tumour weight, respectively, treatment of tumour-implanted mice with AG60 (30 mg kg-1) resulted in a 96% decrease in tumour weight, relative to control, 14 days after tumour-cell implantation. Dose-related inhibition in tumour growth rate was also observed in animals treated with AG60, with maximum (65%) inhibition noted at a dose of 30 mg kg-1 (ED50 23 mg kg-1). Suppression of body weight increase and elevated plasma glucose levels by acriflavine or AG60 indicated that glucose utilization might be impaired. The anti-tumour effect of AG60 was also determined in CDF1 mice intraperitoneally implanted with Ehrlich ascitic tumour. Ehrlich ascitic tumour proliferation was completely suppressed by AG60 (30 mg kg-1, i.p.). Microscopic analyses of intraperitoneal touch-prints revealed that AG60 was more effective in suppressing tumour proliferation than acriflavine alone. Fluorescent microscopic examinations demonstrated that acriflavine avidly bound with Yac-1 cell plasma membrane, leading to morphological changes in the cells, such as bleb formations, swelling and ballooning. The time-related changes in tumour cell morphology by acriflavine or AG60 might represent energy depletion, followed by osmotic lysis as a result of cationic influx. Enhanced anti-tumour activity of acriflavine in combination with guanosine might be explained by the blocking of nutrient transport through selective acriflavine binding with plasma membrane and by concomitant guanosine perturbation of cellular ATP production. This study demonstrates that guanosine enhances the anti-tumour effects of acriflavine against a variety of cancer cells without serious adverse effects, providing a preclinical basis for potential application of this combination against cancer proliferation.